CN104630201A - Rapid DNA extraction kit of medicinal material - Google Patents
Rapid DNA extraction kit of medicinal material Download PDFInfo
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- CN104630201A CN104630201A CN201310548185.5A CN201310548185A CN104630201A CN 104630201 A CN104630201 A CN 104630201A CN 201310548185 A CN201310548185 A CN 201310548185A CN 104630201 A CN104630201 A CN 104630201A
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Abstract
The invention discloses a rapid DNA extraction kit of a medicinal material, belongs to the field of the molecular biology, and relates to the traditional Chinese medicine and traditional Chinese medicinal material identification field. The kit comprises a solution A, a solution B, a reagent C and a user manual; and the solution A is composed of a solute triton-100 and a solvent water, the concentration of the solute is 1%, and the final concentration of the solute in the solution A 1%. The solution B is composed of 1M of a solute Tris-HCl and the solvent water, the concentration of the Tris-HCl in the solution B is 1M, the pH value of the solution B is 8.0, the concentration of the solute is 0.1M, and the final solution of the solute in the solution B is 0.1M. The reagent C is composed of NaOH and polyvinylpyrrolidone (PVP, M=40), the final concentration of NaOH in the solution A is 0.5M, and the final concentration of the PVP in the solution A is 1%. The kit is a medicinal material genome DNA extraction product developed for rapidly discriminating the medicinal material, and can be used for rapidly extracting plant and animal medicinal material genome DNA. The kit has the advantages of few operation steps, fast extraction speed, low price, no need of a large device, large-scale high-flux extraction and the like, and is very suitable for rapid detection and identification of the medicinal material DNA.
Description
Technical field
The present invention relates to Chinese medicine and Materia Medica Identification field, particularly a kind of medicinal material Fast DNA extraction test kit.Belong to technical field of molecular biology
Background technology
The extraction of DNA is for a long time step consuming time, the most loaded down with trivial details in DNA analysis technology experiment always, seriously restricts using and promoting of molecular assay method.For reaching the object of DNA rapid extraction, investigator successively proposes the extraction for DNA of plants such as ROSE method, magnetic bead absorption method, Tris-EDTA extraction method.Although these methods shorten the time of DNA extraction, still employ the steps such as heating, high speed centrifugation, precipitation due to it, still cannot realize the object of fast and convenient extraction DNA.Wherein DNA alkaline lysis is considered to a kind of effective DNA rapid extracting method, has been widely used in wheat, corn, peanut, paddy rice etc., in marker assisted selection, transgenic plant qualification, SSR qualification etc., serve vital role.DNA alkaline lysis passes through sodium hydroxide or the potassium hydroxide treatment vegetable material of 0.2-1.0M, and lysing cell also obtains DNA, has the advantages that method is simple, operation steps is few, do not need to use the toxic reagents such as phenol.But because vegetable material difference is huge, be difficult to find general alkaline lysis agent prescription, yet there are no the report using alkaline lysis method of extracting Chinese medicinal materials in addition.The present invention establishes the neutralization reagent rapid extraction medicinal material DNA utilizing alkaline lysis to add new additive agent Triton X-100 (TritonX-100) and applicable rapid extraction medicinal material DNA.
Summary of the invention
The application's object is to provide a kind of method of medicinal material Fast DNA extraction, this DNA extraction method operation steps is few, extraction rate is fast, cheap, do not need large-scale instrument, can on a large scale high-throughput extract, may be used for the rapid extraction of plant, animal drug genomic dna.
1. a medicinal material Fast DNA extraction test kit, this test kit comprises Extraction buffer, neutralization buffer, service manual, wherein said Extraction buffer comprises solution A and reagent C, wherein solution A is made up of solute Triton X-100 (tritonX-100) and aqueous solvent, reagent C is made up of NaOH and polyvinylpyrrolidone (PVP, M=40); Described neutralization buffer comprises solution B, and described solution B is made up of solute 1M Tris-HCl and aqueous solvent.
2. the Extraction buffer described in comprises solution A and reagent C, and described solution A is made up of solute Triton X-100 (tritonX-100) and aqueous solvent, described solute and in solution A ultimate density be 1%; Described reagent C is made up of NaOH and polyvinylpyrrolidone (PVP, M=40), and NaOH ultimate density in solution A is 0.5M, PVP is 1%.
3. the neutralization buffer described in comprises solution B, and described solution B is made up of solute 1M Tris-HCl and aqueous solvent, and in solution B, the concentration of Tris-HCL is 1M, pH=8.0; Described solute and the ultimate density in solution B thereof are 0.1M.
4. the test kit described in, it is characterized in that its rapid detection being highly suitable for the DNA of medicinal material and qualification, the DNA fragmentation length using this test kit to obtain is generally at about 1Kb, be applicable to the amplified reaction carrying out about 500bp fragment, DNA concentration is 354.87 scholar 154.96ng/ μ L, and A260/A230 value is 1.17 scholars 0.24.
5. a method of rapid extraction medicinal material DNA, specifically comprises the steps:
(1) reagent C is dissolved in solution A,
(2) Extraction buffer and medicinal powder are fully mixed, add neutralization buffer, mixing, centrifugal, get supernatant liquor,
(3) supernatant liquor of neutralization buffer and step (2) gained is mixed, centrifugal, get the medicinal material DNA that supernatant liquor is rapid extraction.
6. the method for the rapid extraction medicinal material DNA according to 5, is characterized in that reagent C should be dissolved in solution A by Extraction buffer, uses until completely dissolved, validity period is 20 days.
7. the method for the rapid extraction medicinal material DNA according to 5, it is characterized in that the amount of medicinal powder is 10-25mg, the amount adding Extraction buffer is 200 μ L, the amount that step (2) adds neutralization buffer is 800 μ L, and the amount adding neutralization buffer in step (3) is 500 μ L.
8. the method for the rapid extraction medicinal material DNA according to 5, is characterized in that described centrifugal for 12000rpm, centrifugal 1min.
9. the present invention establishes the test kit utilizing alkaline lysis rapid extraction medicinal material DNA, the test kit provided is provided, do not need to prepare any reagent separately, operator can grasp the method for DNA extraction without the need to Special Training, can be used for the extraction of the raw product of Chinese medicinal materials and process of preparing Chinese medicine material DNA, all have good effect for animal and plant medicinal material, the Chinese medicinal materials DNA of extraction can be used for PCR reaction.
Accompanying drawing explanation
Fig. 1: extract snake class medicinal material genomic dna with this product, and utilize universal primer ColI to carry out PCR reaction, get 5 μ L and detect.M is DL2000Marker, N: negative control; 1: Zaocys; 2: mone snake; 3: Elaphe Carinatas; 4: Sinonatrix annularis
Fig. 2: extract plant medicinal material genomic dna with this product, and utilize universal primer psbA-trnH to carry out PCR reaction, get 5 μ L and detect.M:DL2000Marker; N: negative control; 1: root tuber of aromatic turmeric; 2: Silktree Albizzia Bark; 3: Radix Angelicae Sinensis; 4: Japanese Honeysuckle; 5: ginseng; 6: Semen Plantaginis; 7: windproof; 8: Wrinkled Gianthyssop Herb; 9: safflower; 10: kuh-seng; 11: the flower bud of lily magnolia; 12: Fruit of Threeleaf Akebia; 13: the red sage root; 14: Yanhusuo; 15: balloonflower root; 16: Thunberg Fritillary Bulb; 17: the root of herbaceous peony; 18: Tradescantia albiflora; 19: Tuber Fleeceflower Stem; 20: Chinese yam; 21: Radix Codonopsis.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all obtain from pharmacy and commercial sources.
Embodiment 1
1, snake class medicinal material DNA rapid extraction
A kind of medicinal material DNA rapid extraction test kit, comprises Extraction buffer (solution A, reagent C), neutralization buffer (solution B) and service manual.Wherein solution A is the tritonX-100 aqueous solution of 1%, and solution B is the Tris-HCl aqueous solution of 0.1M, and reagent C is NaOH and PVP.Use this test kit to carry out the DNA rapid extraction of snake class medicinal material, step is as follows:
(1) reagent C (0.08gNaOH, 0.04gPVP) is dissolved in solution A (4mL), is made into DNA extraction damping fluid, stand-by after dissolving completely.
(2) get each 25mg of dry medicinal material of Zaocys, mone snake, beautiful rat snake, Sinonatrix annularis, put in 2mL centrifuge tube respectively, use ball milling instrument to be about 5min after numbering and medicinal material is pulverized, stand-by.
(3) respectively to adding 200 μ LDNA Extraction buffers in 4 increment product, vortex oscillation fully mixes.
(4) respectively to adding 800 μ L solution B in 4 increment product, vortex oscillation fully mixes, the centrifugal 1min of 12000rpm or standing 10min.
(5) get in the new 2mL centrifuge tube of 500 μ L supernatants to, then add 500 μ L solution B respectively, vortex oscillation fully mixes.Preserve or dilute 10-100 for-20 DEG C doubly to react for PCR.
2, augmentation detection
Use universal primer CoII, increase according to following system: cumulative volume 25 μ L, wherein 10 × buffer damping fluid 2.5 μ L, dNTP10mmol.L
-1, 1.0 μ L), each 10 μm of ol.L of positive anti-primer
-10.25 μ L, rTap enzyme 5U. μ L
-10.2 μ L, DN A20ng (1 μ L), sterile purified water 20 μ L.Reaction conditions: after 95 DEG C of denaturation 5min, 95 DEG C of sex change 30S, 58 DEG C of annealing 45S, 72 DEG C extend 1min, and 35 rear 72 DEG C of extension 5min of circulation, obtain PCR primer.
Get 5 μ L amplified productions, adopt the sepharose of 1%, do reference with D L2000DNA marker, under voltage 100-150V, electrophoresis 10-15 minute, observes under gel imaging system and takes pictures.See Fig. 1.
Embodiment 2
1, plant class medicinal material DNA rapid extraction
(1) reagent C (0.2g NaOH, 0.1g PVP) is dissolved in 10mL solution A (the tritonX-100 aqueous solution of 1%), is made into DNA extraction damping fluid, stand-by after dissolving completely.
(2) each 25mg of dry medicinal material of root tuber of aromatic turmeric, Silktree Albizzia Bark, Radix Angelicae Sinensis, Japanese Honeysuckle, ginseng, Semen Plantaginis, windproof, Wrinkled Gianthyssop Herb, safflower, kuh-seng, the flower bud of lily magnolia, Fruit of Threeleaf Akebia, the red sage root, Yanhusuo, balloonflower root, Thunberg Fritillary Bulb, the root of herbaceous peony, Tradescantia albiflora, Tuber Fleeceflower Stem, Chinese yam, Radix Codonopsis is got, put in 2mL centrifuge tube respectively, use ball milling instrument to be about 5min after numbering medicinal material is pulverized, stand-by.
(3) respectively to adding 200 μ LDNA Extraction buffers in 21 increment product, vortex oscillation fully mixes.
(4) respectively to adding 800 μ L solution B (the Tris-HCl aqueous solution of 0.1M) in 21 increment product, vortex oscillation fully mixes, the centrifugal 1min of 12000rpm or standing 10min.
(5) get in the new 2mL centrifuge tube of 500 μ L supernatants to, then add 500 μ L solution B respectively, vortex oscillation fully mixes.Preserve or dilute 10-100 for-20 DEG C doubly to react for PCR.
2, augmentation detection
Use universal primer psbA-tmH, increase according to following system: cumulative volume 25 μ L, wherein 10 × buffer damping fluid 2.5 μ L, dNTP10mmol.L
-1, 1.0 μ L), each 10 μm of ol.L of positive anti-primer
-10.25 μ L, rTap enzyme 5U. μ L
-10.2 μ L, DNA20ng (1 μ L), sterile purified water 20 μ L.Reaction conditions: after 95 DEG C of denaturation 5min, 95 DEG C of sex change 30S, 56 DEG C of annealing 30S, 72 DEG C extend 1min, and 40 rear 72 DEG C of extension 5min of circulation, obtain PCR primer.
Get 5 μ L amplified productions, adopt the sepharose of 1%, do reference with D L2000DNA marker, under voltage 100-150V, electrophoresis 10-15 minute, observes under gel imaging system and takes pictures.See Fig. 2.
Claims (8)
1. a medicinal material Fast DNA extraction test kit, this test kit comprises Extraction buffer, neutralization buffer, service manual, wherein said Extraction buffer comprises solution A and reagent C, wherein solution A is made up of solute Triton X-100 (tritonX-100) and aqueous solvent, reagent C is made up of NaOH and polyvinylpyrrolidone (PVP, M=40); Described neutralization buffer comprises solution B, and described solution B is made up of solute 1M Tris-HCl and aqueous solvent.
2. test kit according to claim 1, it is characterized in that described Extraction buffer comprises solution A and reagent C, described solution A is made up of solute Triton X-100 (tritonX-100) and aqueous solvent, described solute and in solution A ultimate density be 1%; Described reagent C is made up of NaOH and polyvinylpyrrolidone (PVP, M=40), and NaOH ultimate density in solution A is 0.5M, PVP is 1%.
3. the test kit according to claims 1, it is characterized in that described neutralization buffer comprises solution B, described solution B is made up of solute 1M Tris-HCl and aqueous solvent, and in solution B, the concentration of Tris-HCL is 1M, pH=8.0; Described solute and the ultimate density in solution B thereof are 0.1M.
4. the test kit according to claims 1, it is characterized in that its rapid detection being highly suitable for the DNA of medicinal material and qualification, the DNA fragmentation length using this test kit to obtain is generally at about 1Kb, be applicable to the amplified reaction carrying out about 500bp fragment, DNA concentration is 354.87 ± 154.96ng/ μ L, and A260/A230 value is 1.17 ± 0.24.
5. a method of rapid extraction medicinal material DNA, is characterized in that adopting test kit described in claim any one of claim 1-4 to extract medicinal material DNA, specifically comprises the steps:
(1) reagent C is dissolved in solution A,
(2) Extraction buffer and medicinal powder are fully mixed, add neutralization buffer, mixing, centrifugal, get supernatant liquor,
(3) supernatant liquor of neutralization buffer and step (2) gained is mixed, centrifugal, get the medicinal material DNA that supernatant liquor is rapid extraction.
6. method according to claim 5, is characterized in that reagent C should be dissolved in solution A by Extraction buffer, uses until completely dissolved, validity period is 20 days.
7. method according to claim 5, it is characterized in that the amount of medicinal powder is 10-25mg, the amount adding Extraction buffer is 200 μ L, and the amount that step (2) adds neutralization buffer is 800 μ L, and the amount adding neutralization buffer in step (3) is 500 μ L.
8. method according to claim 5, is characterized in that described centrifugal for 12000rpm, centrifugal 1min.
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| CN201310548185.5A CN104630201A (en) | 2013-11-08 | 2013-11-08 | Rapid DNA extraction kit of medicinal material |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695620A (en) * | 2016-04-27 | 2016-06-22 | 成都中医药大学 | Method for rapidly detecting Chinese caterpillar fungus |
| CN105695460A (en) * | 2016-04-27 | 2016-06-22 | 成都中医药大学 | Kit for rapidly detecting Chinese caterpillar fungus |
| CN106350508A (en) * | 2016-10-17 | 2017-01-25 | 中国医学科学院药用植物研究所 | Extraction method and kit of genome DNA of forest frog's oviduct, and application thereof |
| CN109628313A (en) * | 2019-01-24 | 2019-04-16 | 长春万成生物电子工程有限公司 | A kind of E. coli lysate and its preparation method and application |
| CN110894537A (en) * | 2019-11-07 | 2020-03-20 | 澳门科技大学 | PCR (polymerase chain reaction) primer, kit and method for identifying authenticity of cordyceps sinensis and commercial product thereof |
| CN113322313A (en) * | 2021-06-19 | 2021-08-31 | 长沙理工大学 | Method for rapidly identifying plant genes |
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| CN101575597A (en) * | 2009-05-21 | 2009-11-11 | 中国农业大学 | Kit for quickly extracting plant genome and applications thereof |
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| CN101575597A (en) * | 2009-05-21 | 2009-11-11 | 中国农业大学 | Kit for quickly extracting plant genome and applications thereof |
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| 段中岗等: "适合中药材DNA条形码分析的DNA提取方法的研究", 《中药新药与临床药理》 * |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695620A (en) * | 2016-04-27 | 2016-06-22 | 成都中医药大学 | Method for rapidly detecting Chinese caterpillar fungus |
| CN105695460A (en) * | 2016-04-27 | 2016-06-22 | 成都中医药大学 | Kit for rapidly detecting Chinese caterpillar fungus |
| CN106350508A (en) * | 2016-10-17 | 2017-01-25 | 中国医学科学院药用植物研究所 | Extraction method and kit of genome DNA of forest frog's oviduct, and application thereof |
| CN106350508B (en) * | 2016-10-17 | 2019-01-18 | 中国医学科学院药用植物研究所 | Oviductus Ranae genome DNA extracting method, kit and its application |
| CN109628313A (en) * | 2019-01-24 | 2019-04-16 | 长春万成生物电子工程有限公司 | A kind of E. coli lysate and its preparation method and application |
| CN110894537A (en) * | 2019-11-07 | 2020-03-20 | 澳门科技大学 | PCR (polymerase chain reaction) primer, kit and method for identifying authenticity of cordyceps sinensis and commercial product thereof |
| CN110894537B (en) * | 2019-11-07 | 2023-08-08 | 澳门科技大学 | PCR primer, kit and method for identifying cordyceps sinensis and commodity authenticity thereof |
| CN113322313A (en) * | 2021-06-19 | 2021-08-31 | 长沙理工大学 | Method for rapidly identifying plant genes |
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